JPH07507688A - diphtheria toxin vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] diphtheria toxin vaccine Invention The invention described in this application was published by the Public Health Service from the National Institute of Allergy and Infectious Diseases. Funded at least in part by Bureau grants Al-22021 and Al22848. This was done during financially supported research. The U.S. government has approved several have the right.

The present invention relates to vaccines that protect against diphtheria toxin.

Diphtheria toxin (DT) is produced by the bacterium Corynebacterium diphtheriae It is a protein exotoxin. DT molecules are easily nicked medium polypeptides. as a result of cleavage at residues 190.192 or 193. Two subunits, fragment A (N-terminus- 21K) and fragment B (C-terminus -37K) (Moskaug et al., Biol Chem, 264:15709-15713.1989;C.

11ier et al., Biol Chem, 246:1496-1503.1971) .

Fragment A is the tentacle active portion of DT. It is elongation factor 2 (EF-2) specifically targets protein synthesis factors called NAD-dependent AD (inactivates EF-2 in cells and stops protein synthesis) P-ribose transferase. Fragment B of DT is present in many types of mammalian cells. The specific receptor structure found on the surface of the toxin is modified to bind toxin molecules. It has a receptor binding domain. DT connects to cells via this receptor structure. When combined, receptor/DT1 association leads to receptor-mediated endocytosis. taken up by cells. The second functional region of fragment B is the endocytic DT is translocated through the membrane of the catalytically active vesicle and the catalytically active fragment A is transferred to the cell. There is a movement of release into the side sill of the vacuole. A single fragment A molecule is isolated from a given cell is sufficient to inactivate the protein synthesis machinery in

Immunity to bacterial toxins such as DT may be spontaneous or ablated during the course of infection. Artificially obtained by injection of toxoids in the form of Edited by ier, Bacterial Vaccines, Academic P ress, Orlando, Fl., 1984). Toxoids are traditionally Chemical modification of toxins [e.g. CLing with formalin or formaldehyde] o.

d et al., Brlt, J. Exp, Path, 44:177.1963)]. to protect animals that have been produced and vaccinated against subsequent attack by natural toxins. detoxifies toxins while retaining their antigenicity. An example of chemically inactivated DT is , Michel and Dirkx (Biochem, Biophys, Ac ta491:286-295.1977) (fragment Trp-153 of Trp-A is a modified residue). However, that Toxins chemically modified by sea urchins sometimes lose their attached chemical groups or groups, reducing their activity. It reverts to the sexually toxin form and as a result, using it as a vaccine is poses a potential danger to persons.

Another method of producing toxoids is through the use of genetic technology. Korineba C. diphtherium mutant, CRM-197 (Uchida et al., J.

Biol, Chem, 248:3838-3844, 1973; Uchida et al. , Nature 233:8-11.1971) (CRM is an abbreviation for cross-reacting substance. ) is a natural toxin produced by random mutation that produces an anti-DT immune response. It was shown to contain an enzymatically inactive T protein that is fully compatible with . Co1 1ier et al. (U.S. Pat. No. 4,709,017, incorporated herein by reference) ) is a gene engineered to carry a deletion mutation in Glu-148 of diphtheria toxin. Diphtheria toxin variants are disclosed. Glu-148 is activated by antitropism [3] was initially identified as a sex site residue (Carroll et al., Proc., Nat. l.

Acad, Sci, USA 81:3307.1984; Carroll et al., P roc, Natl, Acad, Sci, USA 82 Near 237, 1985; C (Arroll et al., J. Biol. Chem. 262:8707, 1987). Substitution with Asp, Gln or Set at the site results in enzymatic and cytotoxic activity. is reduced by about 2-3 times, and the spatial (steric) (H[ show that position and chemistry significantly influence these activities ( Cglas et al., J. Bacteriol, 169:4967.1987) , Greenfield et al. (U.S. Pat. No. 4,950,740; (incorporated as a reference) delete the Glu-148 residue or with Asn. and a genetically engineered mutant of DT in which the Aja-158 residue was replaced with cty. Disclosed. DNA sequence and corresponding amino acid sequence of wild-type diphtheria toxin is shown in FIG. 1 (SEQ ID NO: 1).

Summary of the invention Recent approaches to vaccination include mutant ZX, a living organism expressing the gene. Using genetically engineered microorganisms (cells or viruses). Among the vaccinated people, raw Cutin is a vaccine that contains genetically engineered genes encoding toxoids. It allows the genes to multiply, so in theory, over time, the mutate. Such naturally occurring mutations make genetically engineered toxoids toxic. If reversion occurs, illness and/or death of the vaccinated person will result. Applicant is genetic A powerful scavenger of engineered diphtheria toxoid, Colier et al. DT Glu-148 deletion mutant disclosed in Japanese Patent No. 4,709,017) found that there was a small but possibly significant risk of reversion to partial toxicity. are doing. Additionally, Applicants have determined that the antigenicity or stability of the resulting polypeptides has not been exceeded. We have discovered ways to reduce this risk without exposing ourselves to more risks. present invention The toxoids and the DNA encoding them are Glu-1 of Colier et al. has a significantly lower risk of reversion than the 48 deletion mutant and therefore Substantially better for use in live, genetically engineered vaccine cells that can be propagated. This is a good candidate.

The present invention encodes an immunologically cross-reactive form of diphtheria'di1g fragment A. or the naturally occurring diphtheria toxin Va 1-147 and G Codon 17 (E characterized by DNA encoding both fragment A and fragment B. There is. In addition, a codon corresponding to the naturally occurring third amino acid residue of the toxin is missing. or encode amino acid residues that are different from those of the naturally occurring toxin. 1) This third amino acid residue of the naturally occurring toxin can be modified to The presence of the group is essential for full toxin activity of naturally occurring toxins. tertiary amino acid The residue may be in the fragment A portion of diphtheria toxin, in which case The naturally occurring third amino acid residue of the toxin is, for example, GI Y-52, G I Y-79, cly-128, A! a-158 or Glu-162.

The third amino acid residue is found in fragment 8 of diphtheria toxin instead of 1. The third amino acid residue of a naturally occurring toxin may be, for example, Glu-349, A sp-352 or l1e-364. Preferably, Glu 142 is The corresponding Kodoyu does not exist or encodes an amino acid other than Glu. or all codons from Glu-142 to Gju-148 does not exist. ``Naturally existing °'' is shown in Figure 1 (SEQ ID NO: 1) refers to wild type diphtheria toxin having the amino acid sequence. “Perfect” that exists in nature ``Toxin activity'' means 1llllll in 14 cell killing assays as described below. The ability of wild-type diphtheria toxin to attach to, penetrate and kill cells is 1009. , means.

The present invention includes DNA sequences encoding the Fragment A variants described in this application. Also includes vectors (ie, plasmids, phage, and viruses) that contain of the present invention Expression of diphtheria toxoid polypeptide is under the control of a heterologous promoter. and/or the expressed amino acids are linked to a signal sequence. “Atypical The promoter ° is the promoter found in the naturally occurring diphtheria toxin gene. means a promoter region that is not identical to the promoter region of Promoter territory region is the DNA segment 5' of the transcription start site of a gene, where the RNA polypeptide is inserted. Merase binds to genes before transcription begins. Preparation of essentially pure nucleic acids of the invention A product is a preparation containing the nucleic acid of the present invention, in which the nucleic acid encoding diphtheria toxin is naturally present. However, f4 contains substantially no other nucleic acid molecules bound to Corynebacterium. stomach.

The DNA encoding the diphtheria toxoid of the present invention is present in cells or cell groups in the stroma. , preferably B. subtilis, Bacillus bacilli (BCG), Salmonella species, Vibrio cholera, Corynebacterium diphtheriae, Listeri Contained in E. yersiniae, Streptococcus or E. coli cells. Thin Preferably, the cells are capable of expressing a diphtheria toxoid of the invention.

The term “immunological cross-reactivity” used in this application indicates that diphtheria toxoid is retains at least one antigenic determinant common to naturally occurring diphtheria toxins; , as a result, they contain a small number of molecules that have specificity for naturally occurring diphtheria toxins. Each is bound by at least one antibody. “Book” as defined in this application The diphtheria toxoid of the invention is immunologically compatible with naturally occurring diphtheria toxin. means a diphtheria toxoid that reacts differentially and is exemplified or claimed in this application. It retains one of the modifications made. “Immunological interaction of diphtheria toxin fragment A” Differentially reactive ° includes diphtheria toxin polypeptides that immunologically cross-react; It has at least 40% homology with naturally occurring fragment A.

The vaccine of the invention can be any of a variety of D cells or viruses, preferably live vaccines, containing NA or the DNA of the present invention cells, more preferably B. subtilis, BCG, Salmonella sp., Vibrioco Lele, Listeria, Yersiniae, Streptococcus, Corynebacterium zifu seria or E. coli cells. “Live vaccine cells” used in this application are naturally occurring non-toxic live microorganisms that express the immunogen or have low toxicity. or live microorganisms with weakened virulence.

One method for producing the vaccine of the present invention is to use the diphtheria toxoid of the present invention. Cells containing the encoding DNA are cultured under conditions that allow the cells to proliferate. and the cells are suitable for introduction into animals as a live cell vaccine. be. A vaccine is a method that immunizes a mammal, preferably a human, against diphtheria. method (method comprising introducing into a mammal an immunizing amount of a vaccine of the invention). Can be used. A cell-free vaccine harboring DNA encoding the diphtheria toxoid of the present invention One non-restrictive method of administering chloride is biolistic (b. iolisLic) transport [microcells that carry DNA encoding the immunogen of interest] Application of projectile (microprojectile) and coated Miku A delivery method in which the projectile is injected directly into the cells at the end of the recipe (Tan g et al., Nature 356:152-154, 1992: in the references of this application. (incorporation)]. Subsequently, the diphtheria toxoid of the present invention is expressed from DNA. , to stimulate the immune response of the recipe end. elicits an immune response against diphtheria toxin By incorporating an immunogen or a DNA encoding an immunogen, the vaccine of the present invention can be prepared. Cutin is effective against the progression of diphtheria disease and against the bacterium Frynebacterium diphtheriae. immunize against infection by E.

In other embodiments, the invention provides immunological crosslinking of diphtheria toxin fragment A. Polypeptides that are differentially reactive or, preferably, naturally occurring diphtheria Amino acids corresponding to toxin Val-147 and Glu-148 (SEQ ID NO: 1) is present in the polypeptide. It is characterized by polypeptides that are differentially reactive. Preferably, the toxoid is Tyr- Hold 149. Preferably, the naturally occurring third amino acid residue of Toxin 1 is Amino acids that are deleted or different from the naturally occurring third amino acid residue of the toxin The toxin has been modified to encode a residue and is naturally The presence of a third amino acid residue is essential for the full toxin activity of the naturally occurring four elements. Ru. The third amino acid residue is in the fragment A portion of the diphtheria toxin and is preferably are His-21, Glu-22, Lys-39, CI! -52, G I V- 79, c+y-128, Ala-158 or Glu-162, or diphtheria Fragment 8 of atoxin, preferably amino acids Glu-349, As 1)-352 or l1e-364. Furthermore, the polypeptide Glu- 142 is either absent or modified to an amino acid other than Glu. Or, G All amino acids from lu-142 to G l u-1,48 are absent is also possible. Polypeptides are preferably prepared according to the present invention by any suitable method. Any of a variety of cells containing DNA encoding phtheria toxoid can be cultured to produce DNA. It is produced by culturing under row 1'F, which allows for included in the invention is a substantially pure preparation of a polypeptide of the invention. What is substantially pure? According to the invention, at least 5096 (by weight) of tan/(phosphorus) present in the preparation diphtheria toxoid polypeptide. In a preferred embodiment, the preparation At least 759o, more preferably less, of h4 tongue/extract in the product at least 9096, most preferably at least 9996 (by weight) of the diphthene of the present invention. It is a liatoxoid polypeptide.

The vaccine against diphtheria toxin is the diphtheria toxoid polypep of the present invention. made from a composition that includes a compound and an adjuvant. Adjuvant is optional Currently known types of adjuvants, e.g. aluminum salts, bacterial endotoxins, Bacillus Bacillus Lumette-Guerlain (BCG), liposomes, microspheres (suna Specifically, microcapsule polymers used in orally administered vaccines) and fluoride Contains complete or incomplete adjuvant, etc. “Adjus” used in this application "Bant" is a substance that can increase the immunogenicity of an antigen.

The diphtheria toxoid polypeptide of the present invention may be a polysaccharide or another diphtheria toxoid polypeptide. It may also be covalently attached to the polypeptide moiety. This part is polypeptide The diphtheria toxoid polysaccharide of the present invention can also serve as a carrier material for the diphtheria toxoid of the invention. The peptides act as carrier substances for these moieties and preferably reduce the immunogenicity of these moieties. strengthen. A “carrier material” confers stability to the bound molecules and/or Substances that aid in or enhance the delivery or immunogenicity of combined molecules.

The diphtheria toxoid of the present invention binds to another polypeptide through a peptide bond. A fusion polypeptide consisting of the diphtheria toxoid polypeptide of the present invention It is also a part of Preferably, the fusion polypeptide is included in a vaccine and Used to immunize human patients against atoxins. Diphtheria of the present invention The toxoid polypeptide acts as a carrier material for the attached polypeptide and is a preferred Preferably, the immunogenicity of the added polypeptide is enhanced. fusion polypeptide The DNA encoding the can be used directly as a vaccine or can be integrated into cells. , preferably cells capable of expressing the fusion polypeptide (e.g. live vaccine cells) are Used as a vaccine against phtheria toxin. ``Fusion point'' used in this application "ripeptide" means that the DNA encoding the diphtheria toxoid of the present invention is derived from a gene. operatively linked to a second DNA encoding a second polypeptide sequence Refers to a protein molecule produced by expression of hybrid DNA.

“Homology” as applied to this application refers to the sequence identity or sequence identity between two polypeptide molecules. refers to sequence identity between two nucleic acid molecules. the location of both the two compared sequences If a given position is occupied by a subunit of the same amino acid monomer, e.g. Each position in the two polypeptide molecules is occupied by aspartic acid. , then the molecules are homologous at that position. The homology between two sequences is It is a function of the number of matched positions common to the array. For example, 10 in two arrays If 6 of the positions are compatible or homologous, then the two sequences are 60? o homologous be. For example, the amino acid sequences LTVSFR and LPVSAT are 50% homologous. 9 together. In a preferred embodiment of the invention, diphtheria toxin fragment A or immunologically cross-reactive forms of fragment B are naturally occurring, respectively at least 4096, preferably 5096, more preferably or at least 6096 homologs, most preferably at least 8096 homologs.

Applicant intends to administer the toxoid of the invention to humans in the form of a live attenuated vaccine strain. Method for constructing a variant of diphtheria toxin fragment A that is safe to administer to Tadateshi It shows. Use of live vaccine strain immunizes with diphtheria toxoid lJt There are many advantages to doing so. Living creatures multiply in the recipe end and become clowns. Expresses encoded protective protein antigens. A live attenuated vaccine is injected It remains in the recipient for a longer time than polypeptides that can be genetically engineered in 5 in situ. Continuously produces the protein that is produced. Such live vaccines are effective immunization treatments. may require fewer injections or boosters, and are often given orally. It is administered orally and can be used to administer multiple doses of antigen.

For this purpose, Applicants have proposed amino acids near the active site of ADP ribose transferase activity. amino acid, that is, the amino acid on the NH2-terminal side of Glu-148 in FIG. was experimentally deleted or replaced. In doing so, it changes to Glu residual When the key residue Glu-148 is deleted (DT) They determined which amino acid positions restored activity. this recognition In order to reduce the possibility that phenotypic reversion occurs in vivo, Deleting or modifying residues. In this way, the applicant Even in the host, enzymatically dysfunctional We created 21 mutants of atoxin.

In combination with a pharmaceutically suitable carrier to form a vaccine composition to be administered to mammals. Combined? 11 Toxoids exert immunoprotection against diphtheria'tA. live. The toxoid is the DNA encoding the toxoid and its expression. By culturing cells containing a DNA propagator with a DNA that can be is produced.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims. Dew.

Detailed description of the invention We will first explain the drawing inside the cylinder.

drawing Figure 1 shows wild type diphtheria! [Nucleo of DNA encoding (SEQ ID NO: 1) 1 is a representation of the sequence and the corresponding amino acid sequence.

FIG. 2 is a schematic representation of the secondary structure in which Glu-148 exists. The diagram shows DT Dyma (Collier et al., U, S, S, N, No. 07/881,394 3 Incorporated into the references of this application HCh .

et al., Nature 357:216-222.1992), Glu-148 ( E14g) is found to be present in the β-strand, with one residue removed from the loop and the adjacent N Connect this strand with the H2 proximal β strand. Backbone N(-m- ) and carbonyl O (□) atoms.

Glu-148 deletion mutant constructs (DT delta-148 and Research has been carried out on the second possible site of mutation in the protein. two mutations It was found that the activity was partially restored by both: valine. 147 to glutamic acid (two base change), or towards the amino acid end. Deletion of 5 residues (15 nucleotide deletion), which converts Glu-142 to Tyr -149. Therefore, important proteins such as Glu-148 Simply deleting a residue may result in a second-site mutation resulting in partial activity in recombinant toxoids. There is no guarantee that it will not recover.

This suggests that further genetic abnormalities (in order to restore toxicity) may be necessary in DT. (requires sufficient mutations such that it is unlikely to occur naturally) The applicant was urged to do so. First, two amino acid deletions (residues 147.148) It was created at the active site of DT. This mutation alone causes DT to inhibit protein synthesis. The toxicity of the xoid was reduced to 10-4 or less than that of the wild type. Furthermore, only 2 left! +14 In order for 6 to mutate to glutamate and restore detectable activity, the appropriate 3-salt A radical mutation must occur. Second, the isoleucine residue at position 364 Converted to lysine. This residue is located in the translocation domain. and the translocation of DT from the endocytotic vesicle to the side sill. plays an important role in the country. This mutation uniquely affects wild-type DT. In comparison, it produces toxoids that are 500 times more deficient in inhibiting protein synthesis. Ru.

364 lysine mutates to isoleucine, and to restore toxicity, the appropriate Two base mutations must occur.

Experiment information Method Preparation and Analysis of Mutant Diphtheria Toxoids Deletions and substitutions were made as described below. Oligonucleotide characteristics of sea urchin diphtheria toxin fragment A (DTA) gene Created by aberrant mutagenesis. Subsequently, the mutant gene was isolated using standard methods. E., colt or any other standard expression system, as described below. extracts were assayed for NAD:EF-2 ADP ribose transferase activity; DT-specific proteins are examined by Western blot analysis.

Example A plasmid encoding the F2 fragment of DT, pBRptacBamHIA TGF2 was derived from Greenfield et al. (Greenfield et al., PNAS, 80 : 6853-6857.1983). DT F2 flag The fragment contains the naturally occurring DT leader sequence, all Fragment A, and Fragment A. contains the N-terminal 189 amino acid residues of B, such that the final construct has SEQ ID NO. Contains amino acids 1-382 of 1.

Plasmid F2 was digested with BamHI and C1al. Obtained 949 base pairs The fragment was treated with BamHI and Accl restriction enzymes. 19 to obtain M13mp19-F2. Spanning the F2 translation start codon Ndel restriction site, and a translation stop codon at Arg-192 of F2. rS et al. (Nucleic Ac1ds Res, 16:791, 198g) M13mp19-DTA was created by the site-directed mutagenesis method described in I got it. The 968 base pair Ndel-Hindlll block of M13mp19-DTA The fragment was treated with Ndel and Hindll restriction enzymes in pT7-7 ( Tab.

r, Current Protocols in Molecular Bi.

1ogy, edited by Au5ube I et al., Greene, Wi Iey-1nter science, New York, 1991, pages 16.2.1-1 6゜2.11) and clone the resulting plasmid pT7-DTA Each of the DTA site-directed mutation constructs shown in Table 1 was used as a vector. This was prepared. All site-directed mutants are M13mp19-DTAT type DN A and appropriate oligonucleotides. for appropriate active site mutations. 539 base pair Apa l-Ba I 1 restriction of Tagaru Ml 3mp19-DTA The fragment was ligated with Apal and Ba1l restricted pT7-DTA and the competent nt E, c.

1i BL21 (DE3) (Studier et al., J. Mo1. Biol, 1 89:113.1986) was used for transformation. Luria the transformant Cultured overnight in broth (100 μg/ml ampicillin) and M9 minimal medium (10 μg/ml ampicillin). Diluted to 1150 in 0 μg/ml ampicillin) and incubated until ODl, 0. The cells were induced using 1 mVIPTG for 3 hours, and centrifuged (3000xg, 5 minutes). collected. Cell pellets (10mM), 1mM EDTA, pH 8,0 ( Resuspend in 1/30 volume of TE) + 5mM CaC, 15mM MgCl2, and freeze. Conclusion 2ゝ Repeat thawing three times and add O, 1 mg/m + lysozyme and 1 μg/ml DNA Incubate with enzyme I for 15 minutes and centrifuge (10,000 x g, 10 minutes). ) to remove impurities as previously described (Douglas et al. J, Bacteri.

1.169; 4967.1987) I salted it. Subsequently, DTA protein was measured by Western blot analysis and A DP ribose transferase activity was determined as described (Tweten et al., J. Bio. l.

Chem, 260:10392, 1985) assayed.

full length diphtheria toxin Delta 147.148; 3641 > x (and Delta 146-148; 3 Construction of 641>K PT7-DTAT router 147.148 and PT7-DTAT router 146-14 8 was digested with Apal and Mscl. 539 bp Ap spanning each active site deletion The al-Mscl fragment was extracted from a 1-6 agarose gel *#L, ApaI.

separately ligated to Mscl-digested ptac DT 5er148;3641>K. , ptacDT delta 147.148:3641>K and ptacDT delta 146-148; 3641>K was obtained. Competent E using 6 plasmids, E. coli TG-1 was transformed. Transformants were added to Luria broth + 100μg/ ml Ambilicillin (L-amp) overnight and diluted to 1750 with L-amp. diluted, cultured to ODl 0, induced with IPTG for 3 hours, and centrifuged (3000xg, 5 minutes). Cell pellets were washed with 10mM Tris, 1mM EDTA.

pH 8,0 (TE) + 5mM CaCl2.5mM MgCl2 1/30 Resuspend in volume, freeze and thaw 3 times, add O, 1 mg/m + lysozyme and Incubate for 15 minutes with 1 μg/ml DNase 1 and centrifuge (10,000 QXg, 1.0 min) to the G-25 Cefazo Nox cited above. Desalination was carried out using G-50 Cefazo Nox in the same manner as the desalination using G-50 Cefazo Nox.

result After deletion of Glu-1,48C Table 1, mutation 1), the resulting DTA mutant Heterogeneous NAD: EF-2 ADP ribose transferase activity cannot be detected ( 10-4 of wild-type DTA). Va l-1 by Glu residue When combined with the 47 substitution, this deletion produced a product with 6% of wild-type activity. (Table 1, mutation 7). In contrast, in combination with the Glu mutation of Tyr-149 Deletion of Glu-148 (Table 1, mutation 12) resulted in an inactive product.

A longer deletion extending from Glu-148 to residue 144 to the NH2 terminus ( Table 1, mutations 2-5) yielded products with no detectable ADP ribosylation activity.

However, this system containing the deletion of residues M143-148 (including both terminal residues) The following construct (Table 1, mutation 6) has detectable activity (0.6% of wild type) produced protein. In mutation 6, unlike mutations 1-5, adjacent to the deletion (1 combination (mutations 1-5) ranges from 0.696 to 9% of wild type DTA activity was observed.

Completed with deletion of specific active site and addition of membrane translocation domain modification Full-length diphtheria toxin constructs were also evaluated for overall protein stability. was valued. Full-length diphtheria toxin construct (delta 147.148; 3641>K and Delta 146-148; 3641>K) Both Western Pro Software A full-length tongue, with few degradation products, was revealed by the This suggests that the structural integrity of the protein is preserved.

The result of these active site mutations is the localization of the NH2-proximal flank of Glu-148. The repeptide is more flexible and tightly fixed than the local peptide on the proximal side of C0OH. Consistent with the model that it is not. Collie X-ray crystal of DT dimer r et al., U, S, S, N, No. 07/881,394) lend support to this model. I can do it. The Glu-148 residue forms an antiparallel β-sea bordering the active site cleft. NH2 proximal β chain (Fig. 2), which is present in the Glu-148 β chain and adjacent to the Glu-148 β chain. By removing only one residue from the large 10-residue loop (residues 137-146) do.

Polypeptide of 4 residues immediately after Glu-148) (Tsukubone (residues 149- 152) with hydrogen bonds typical of antiparallel β-sheets and other folding interactions. Together, this bond firmly fixes this region of the polypeptide within the protein. .

These results explain two separate genetic changes, one involving a substitution and the other and additional deletions, each with enzymatically inactive diphtheria toxin activity. Reversion of site deletion mutants to a partially toxic state! It is Noh. of recovered activity Levels are in all cases less than 10% of wild type, but the protein is not produced. This is clearly a concern if it is expressed in vivo by a vaccine. Val- The substitution of Glu at 147 changes Val:lton (GTT) to Glu codon (GAA or GAG) = Which of the two J-capable base pair transversions It can also happen. In contrast, the Val-147 and Glu-148 codons Deletion of both Don leaves 5er-146 near Tyr-149; Ser AG A C codon cannot be converted to a Glu codon without changing all three nucleotides. Therefore, this particular 6-nucleotide deletion mutant is a mutation with some recovery of activity. The risk of returning to the body is the risk of returning a variant lacking only the Glu-148 codon. (probably <10-10/cell/generation).

Additionally, active site deletion and another independent abnormality (W1 translocation dysfunction) All) The side effects of diphtheria toxoid in the genes that cause both are at risk of reversion to toxicity. further reduce risk. DT Delta 147.148 combined with 3641>K or delta 146-148, the appropriate five to restore detectable toxicity. Requires base mutations (3 residues, !!146 or 145 and 2 residues 364) Essential.

This recombinant toxoid, DTdelta 147.148; 3641>K, was cloned. ): expressed in coli to determine overall protein stability and riboprotein stability. Transferase activity was evaluated. Western blot analysis is used to analyze single samples with few degradation products. Reveals the full-length protein, completeness of protein stability and overall structure This suggests that the sex is maintained. As expected, recombinant toxoids There was little or no activity (less than 10-4 of the wild type toxin).

immunogenicity We confirmed that the mutant protein produced as described above lacks detectable enzymatic activity. After identification, the mutants were subsequently analyzed for immunogenicity as follows: guinea pig (or other species naturally susceptible to the cell-killing effects of diphtheria toxin): Immunize with the recombinant toxoid of the invention according to the protocol below: 1-50 μg of recombinant toxoid suspended in 50-100 μl of total adjuvant On day 0.12 and day 240, guinea pigs were injected subcutaneously. Next, naturally occurring di Blood samples are tested for reactivity to phtheria toxin by serial dilution. Assay for antitoxin antibodies. To find out if antibodies protect, Those who received a sufficiently high dose of toxoid to induce xoid formation Test for immunity with wild-type diphtheria toxin. elicit a positive response in the above assay. Those toxoids of the invention that emit are desirable candidates for incorporation into live vaccines. be.

Candidate vaccine is injected into DT-susceptible animals and incubated for 2-3 months. After the immunization period, to determine the extent of immunity, a) a lethal dose of naturally occurring or b) Repeated continuous administration of naturally occurring DT to test the immunity of the animal. Suitable cells that have been genetically engineered to express the toxoids of the invention by Live vaccine microorganisms (cells or viruses) were tested.

Preparation and Use of DNA Encoding Diphtheria Toxoid Diphtheria of the Invention DNA sequences encoding toxoids can be expressed in prokaryotic host cells by standard methods. Wear. The DNA encoding the diphtheria toxoid of the present invention can be expressed in a prokaryotic host. carried by a vector operably linked with control signals capable of Ru. If desired, the coding sequence can be expressed in the periplasmic space of the host cell at its 5' end. Coding any known signal sequence capable of effecting secretion of a protein. contains sequences that encode the protein, thereby facilitating protein recovery.

For example, a vector expressing a diphtheria toxoid of the invention or a vector expressing a diphtheria toxoid of the invention or The fusion protein containing the polypeptide (i) functions in E. coli. Reproduction starting point obtained from lasmid pBR322; (ii) same < pBR322? a selectable tetracycline resistance gene obtained from; (iii) a transcription termination region; For example, termination of E. coli trp operon (transcriptional read-through is caused by trp promo (placed at the end of the tetracycline resistance gene to prevent entry into the protein region) :(iV) Transcriptional promoter, such as trp operon promoter or diphte riatoxin promoter; (V) protein-encoding promoter of the present invention: and (v) i) Transcription terminator-1 e.g. E. coli riboma RNA DrnB) TIT2 sequence from the locus. Used in the synthesis of carrier molecule arrays and DNA sequences. methods, construction of fusion genes, and appropriate vectors and expression systems are all within the skill of the art. It is well known. Similar expression systems can be used to generate fused or non-fused polypeptides ( ie for the expression of the polypeptide of the invention only). this These treatments are examples of, but not limited to, the methods of the present invention.

The most frequently used progenitors are represented by various strains of Colt. deer However, other microbial strains can also be used, such as C. diphtheriae. Reopening Starting points, selectable markers, and control sequences obtained from species compatible with the microbial host. A plasmid vector containing the sequence is used. For example, Bolivar et al. 2:95.1977), a derivative of pBR322 was used. three naturally occurring plasmids (two isolated from Salmonella sp. using the fragment obtained from E. coli (isolated from E. coli). can be transformed. pBR322 is ampicillin and tetracycline resistant. containing the gene and resulting in the construction of the desired expression vector, either maintained or disrupted. provides multiple selectable markers to be used. Commonly used prokaryotic expression control Sequences (also referred to as "regulators") are herein referred to as liposome binding site sequences. Both are determined to contain a promoter for transcription initiation that selectively activates the operator 6. be justified. The promoter commonly used to direct protein expression is β- Lactamase (beninlinase), lactose (lac) (Chang et al., Na ture 198:1056, 1977) and tryptophan (trp) pro- Motor system (Goeddel et al., Nucl, Ac1ds Res, 8+4057 ．． (1980) and the PL promoter and N-gene r link obtained from lambda. Posome binding site (Shimatake et al., Nature 292+128°1 981). Examples of microbial strains, vectors, and associated regulatory sequences are included for illustrative purposes. are shown in this application, but are not intended to limit this application.

Preparation and use of polypeptide vaccine The mutant diphtheria toxoid of the present invention is Expressed in any known protein expression system and subsequently purified by standard methods be done. For example, the diphtheria toxoid of the present invention can be synthesized by organic chemical synthesis. , or can be produced as a biosynthetic polypeptide. Q-mechanical chemical synthesis is performed using conventional automated processes. It can be carried out by peptide synthesis methods or by traditional organic compounding techniques.

Those trained in the art will be familiar with conventional methods of protein isolation, such as precipitation, chroma methods such as, but not limited to, totography, immunoadsorption, or affinity techniques. can be used to purify the diphtheria toxoid polypeptides of the invention. present invention cells of a microbial strain genetically engineered to express diphtheria toxoid, or The polypeptide can be purified from the cell culture medium or from the culture medium of the cells.

The purified polypeptide can be used as an adjuvant to increase the immunogenicity of the toxoid [e.g. For example, aluminum salts, bacterial endotoxins or weakly quaternized bacterial strains (e.g. BCG or Vol. Deterra vertussis), attenuated viruses, liposomes, microspheres, and Freund's complete or incomplete adjuvant] and/or further toxo ids or killed or weakened vaccine organisms (to form multivalent vaccines) ) with a suitable carrier (such as saline). Next, that's it. Such vaccines can be delivered by any acceptable method, including oral, parenteral, transdermal, and transmucosal delivery; administration to human subjects, including but not limited to. Administration of micropheres or in sustained release formulations using biodegradable and biocompatible polymers such as micelles, by in situ delivery using gels, or liposomes, or by transgenic ! ! [For example, Tang et al. (Nature 356:152-154, 1992; directly into the patient's cells as described in the incorporated references of this application). by biolistic administration of Ming DNA].

Preparation and Use of Live Recombinant Vaccines Suitable live carrier organisms include BCG, monkey Monera species, Vibrio cholerae, Streptococcus, Listeria and Erunni Contains weakly tetramorphic microorganisms such as E. The DNA of the invention was prepared using standard methods (Sambroo k et al. Molecular Cloning: A Laboratory Ma nual, Co1d Spring Harbor Lab, Press, New York, 1989) to stably transfect such microbial strains. and subsequently introduced into the patient, eg, orally or by oral administration. to a cheater Once introduced, the bacterium multiplies and expresses a variant form of diphtheria toxin in the patient, causing the disease to become infected. subjects to maintain protective levels of antibodies against the mutant toxin. In a similar way, Adenovirus, herpesvirus, vaccinia virus, polio, fowlpox virus Attenuated animal viruses such as P. spp. or attenuated eukaryotic parasites such as R. Even living organisms can be used as carrier organisms. Genetically engineer the mutant DNA of the present invention into the genome of any suitable virus, followed by standard methods. The virus is introduced into humans and those who receive vaccinations. The live vaccine of the present invention, for example Approximately 10'-108 organisms/dose or protective level sufficient to stably induce antivenom. Administer a sufficient dose. The actual dosage of such vaccines remains in the field of vaccine technology. easily determined by a person of ordinary skill in the art.

Cell killing assay The standard method for assaying the toxicity of diphtheria toxin variants is the diphtheria toxin receptor. cell line (e.g. viro or B5C1 cells) that A phtheria toxin sensitive tissue culture cell line is used. cells with a known amount of mutant diphteate with liatoxin XN supplement or with naturally occurring diphtheria toxin (positive (as a control) or treated with orchid serum albumin (as a negative control) . After incubation, perform a survival assay by counting the surviving colonies ( Yama i z um t, M, et al. Ce1l 15:245-250.19 78).

Alternatively, the degree of cell death can be determined by determining the degree of inhibition of protein synthesis. It can be determined by Incubation with one of the diphtheria toxin samples listed above. After lysis, radiolabeled amino acids (e.g. ['C]Leu) were added to the cell culture. In addition to the growth medium, TCA-precipitated protein scintillator reduces de novo protein synthesis. 1111 is determined by tion counting. Such methods are routine and known to those skilled in the art. It is being

Other embodiments Other embodiments are within the scope of the claims below. For example, Glu-142 and 1 : Diphtheria toxin fragment lacking Val-147 and Glu-148 mutant form of client A, or Glu-142 to Glu-148 (including amino acids at both ends). It is possible to produce a mutant form of diphtheria toxin fragment A that lacks all residues of Wear. Such deletion mutants can be generated by site-directed mutagenesis (5ayers et al., supra). and analyzed for enzymatic properties and immunogenicity as described above. diphtheria poison Other amino acid residues that have been shown to be lacking in natural biological activity are: Residues of fragment A portion of diphtheria toxin, H45-21, Glu-22, L ys-39, Gly-52, GI Y-79, Gly-128, Ala-158 and Gly-162 and residues of fragment 8, Glu-349, As p-352, and 1ie-364. Va l-147 and Glu-1 In addition to missing both of the 48 IAMs, there are mutations missing one or more of these IAMs. Variants are known to those trained in the art [IF site-directed mutagenesis It can be made by a method.

array list (1) General information 2 (i) Applicant: R, John Co11ierKevin P, K11lee n John J, Mekalanos (1. Title of the invention, diphtheria toxin vaccine (iii) Number of sequences: 1 (1v) Address: (A) Pass; Person: Fish & Richardson (B) a: 225 F ranklin 5tree(C) City: Boston (D) State: Massachusetts (E) Country: U, S, A.

(F) ZIP code: 02110-2804 (V) Computer and O5 name Front and model: (A) Disk: 3.5 inch disk, 1.44Mb (B) Device : IBM PS/2 Model 502 or 55SX (C) O3: IBM P, C , DOS () (-Jiun 3.-30) (D) Software: Word Perfect (/ <- John 5.0) (vl) This application data: (A) Application number: (B) Application opening.

(C) Classification/ (v i i) Prior application data: (A) Application number: (B) Application opening/ (v　i　i　i) Information about agents, etc.

(A) Name: Janis K, Fraser (B) Registration number: 34,819 (C) Processing number: 00246/137001 (ix) Communication information/ (A) Telephone: (617) 542-5070 (B) Telephone Fax: (617) ) 542-8906 (C) Telex: 200154 (2) Information regarding sequence number 1 (l-) Characteristics of array・ (Δ) Length・1942 (B) type: Nucleic acid (C) Number of strands: 2 strands (D) Topology/linear (xl) Array: Sequence number = 1 165 1i0175 C Ding ＾ C Ding ACCGAC? ATTCC’rGCAkAGCTGGACGτTAA ? AAG Ding CCAAGA (”rllLsu L@u Pro Ding hr 11m Pro Gly Lys Leu ＾＠p Val ＾sn Lys Sat Lys Ding hr4ko S 440 44% c″　0′　Ri! Seven go　U　8=80＞ao　a＞　aa　C)Q　gtn gtn-+Kiwaki Ma Kidney and ■ Dazzling 03 Pe>niko υkoO ■ 0ko low (5C5(JtJ　(J--ber　ao　u》 Yo 3 Pregnancy 3 Mouth 3 Pregnancy 5 i Tobuki ÷ ＜＞　E-1-H＞　t-1Φ　Ro　octn(5Qh　C5＞　uc:)　 E-IQI (JQI CJCI-+go). ar-(JQI　＜Ha＞　(!l＞　＜-１■　u＄Mozo ■Ht GI 81 Hoyo 8 Ashizuki 8 = Hoyo a < Ill: F-I C3C5-1a ohcn C5C5be-0F-I CJr6 Quuq)-1m h-hr rJ? 111 t)s hOJ-CJs hc+10>0>oc<x- C5g+: o>1 thorn story F, 5 C5 beat 3I ha, ao (J, -1 (J> Q Ko Oo Ko Beto ← - < B (5LI F 41'll C5-Bake <, -+ hm hma<<<Ll> oo oacv LIC5a> ro 8 kan and; shi gikomu = 17 mu 8 pilgrimage 50C5FI C5>C:)<CJ:e)l(1)? IJN UL ( JC53; o, 2z g, 2 3; 65 g; 2. H；　u；■　Mi5　 birth crazy yo 3 l ji δ ji sa eyes nigar yo 3 beats ’5 E embryo yo 3 ni tsume gokoto Φ hm o: I Pe SJI/I u-beko toko rot; rotto Φ C) J knee 1 ocu ←Φ トΦ 〇-←Q, a<　) 4- Betohe Bekoro - 〇-0 Bemi 5 Ji y Nigu Trigram Keikyou Mutsu Nigu U Eavesmoto F to Thatched Thorn l Yo3 Yo Kanagi Gosani Mother 2 Fang Ugi Yo3 Miko Yo3 Kujira ■ H1 Road Kuchibana B, oa story ■ Niragi Grain E Nibaru imitation Kuchibara hit; Takumi Yo3 ■　■i；　8:　　　　　■　　8 　Goyo　4　和訳　（J＞　u＜　C J＜　＜HCJQlri　g3　υ工←−＝　g　鴬　＝　　　　　　　　　::　： Enemy = 0 two two two = = Night Sleep Capture Ko Usu C), -4hu IIC@t-ttoowhri O-f4m C) J: [- 1, -1 (5F-1a) υl! l　F-IQI　CJ(DO〉pe?beh4 ua size t ψ 0--'5 4 pieces 8 pits 7-Niro gourd: shi included 8 CIL+0c5tJ<-y CJ tJ-L) (j (51:<tlJ v+■　Sleep embryo　Eu　Mitsu　Mi　I foot f　Sh==;　foot attendant　8;called　E α Foot f Si-Pi Sl Mother Yo Hon H; H Ni I Kuni 3B Elbow s iji F;,, Qa F E# E l;, #riδ own S Kenichi ■ Testimony Yo3 Poetry EuFIG, 2 Continuation of front page (51) Int, C1, 6 identification symbol Internal reference number C07H21104B 8615-4CCO7K 14/34 8318-4HC12N 1/21 8 828-4B C12P 21102 C9282-4B//(C12N 1/21 C12RIJ9) (C12P 21102 C12R1:19) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE; IT, LU, MC, NL, PT, SE), AtJ, BB, BG, BR, CA, CZ, FI, HU, JP, KP, KR, LK, MGI (72) Inventor: Mechananos Joan, Massachusetts, United States Cambridge Fresh Bond Road

Claims (29)

[Claims] 1. Val-147 of the naturally occurring diphtheria toxin (Figure 1; SEQ ID NO: 1) and diphtheria toxin phthalate, in which the codons corresponding to GIu-148 and GIu-148 are not present in the DNA. DNA encoding an immunologically cross-reactive form of fragment A. 2. the DNA encodes part or all of diphtheria toxin fragment B; The DNA according to claim 1, further comprising a nucleic acid sequence. 3. There is no codon corresponding to GIu-142 (Figure 1: SEQ ID NO: 1), or modified to encode an amino acid other than Glu. DNA. 4. Codons from Glu-142 to Glu-148 (Figure 1: SEQ ID NO: 1) 4. The DNA according to claim 3, wherein all of the DNA is absent. 5. A codon corresponding to the third amino acid residue of the naturally occurring toxin is deleted. or a different amino acid residue from said third amino acid residue of said naturally occurring toxin. The third of the naturally occurring toxins has been modified to encode an acid residue. The presence of amino acid residues is essential for the full toxin activity of the naturally occurring toxin. The DNA according to claim 1. 6. Claim in which the third amino acid residue is in the fragment A portion of diphtheria toxin. The DNA described in 5. 7. The third amino acid residue of the naturally occurring toxin is His-21, Glu- 22, Lys-39, Gly-52, Gly-79, Gly-128, or Gly The DNA according to claim 6, which is lu-162 (Figure 1: SEQ ID NO: 1). 8. Claim in which the third amino acid residue is in the fragment B portion of diphtheria toxin. The DNA described in 5. 9. The third amino acid residue of the naturally occurring toxin is Glu-349, Asp 9. The DNA according to claim 8, which is -352 or 11e-364. 10. The DNA encodes the naturally occurring diphtheria toxin Tyr-149. The DNA according to claim 1, comprising a codon. 11. A polypeptide encoded by the DNA according to claim 1. 12. 12. A substantially pure preparation of the polypeptide of claim 11. 13. A cell containing the DNA according to claim 1. 14. The cells are B. subtilis, BCG, Salmonella species, Vibrio cholerae, Li Stelliae, Yersiniae, Streptococcus, Corynebacterium diphuselia, Or E. 14. The cell according to claim 13, which is an E. coli cell. 15. A vaccine comprising the DNA according to claim 1. 16. A vaccine comprising the cell according to claim 13. 17. A composition comprising the polypeptide of claim 11 and an adjuvant. 18. The cell according to claim 13 is cultured under conditions that allow expression of the DNA. A method of making a polypeptide, including. 19. The method comprises culturing the cells of claim 13 under conditions that allow proliferation of said cells. introducing said cells into an animal as a live cell vaccine, A method of manufacturing a vaccine that is suitable for. 20. the polypeptide is covalently linked to a polysaccharide or a second polypeptide-containing half; 12. The polypeptide of claim 11, wherein the polypeptide is attached with 21. 21. The polypeptide of claim 20, wherein said half comprises a carrier material. 22. 12. The polypeptide of claim 11, which is linked to the second polypeptide by a peptide bond. A fusion polypeptide containing a polypeptide. 23. A fusion polypeptide according to claim 22 or encoding said fusion polypeptide. Vaccines containing DNA that 24. A DNA encoding the fusion polypeptide according to claim 22. 25. A cell comprising the DNA according to claim 24. 26. 21. The polypeptide of claim 20, wherein said polypeptide serves as a carrier material for said half. peptide. 27. Claim 2, wherein said polypeptide acts as a carrier material for said second polypeptide. Polypeptide according to 2. 28. 27. The polypeptide of claim 26, wherein said carrier material enhances the immunogenicity of said half. Chido. 29. 28. The carrier material enhances the immunogenicity of the second polypeptide. polypeptides.